The long term goal of this study is to determine mechanisms regulating mineralization in skeletal tissues. Physiological mineralization occurs in growth plate cartilage, bone and teeth, and is restricted to specific sites. Uncontrolled (pathological) mineralization has severe consequences and leads to morbidity and mortality. Therefore, an understanding of the regulatory mechanisms of mineralization is of crucial importance. During our last funding period we made major progress in understanding the role of calcium homeostasis in the regulation of mineralization. Calcium influx mediated through annexin channels regulates the expression of mineralization-related genes, and the release of mineralization-competent matrix vesicles. Interestingly, overexpression of annexin V resulted not only in increased mineralization but also upregulation of ank gene expression. Ank is a newly discovered protein which transports intracellular pyrophosphate (PPi) to the extracellular millieu. Human mutations in Ank are associated with extensive mineralization in articular cartilage and bone. Our preliminary findings reveal that high expression of Ank in mineralizing growth plate cartilage results in increased extracellular PPi. Increased extracellular PPi lead to upregulation of APase expression and activity, and subsequent mineralization. These findings lead to one hypothesis that Ank is a positive physiological regulator of mineralization by regulating PPi homeostasis and APase expression and activities. In Aims 1 and 2 we propose to determine how Ank regulates the mineralization process and the expression and activities of APase. In Aim 3 we propose to determine the mechanisms regulating ank gene expression during or just before the initiation of the mineralization process. This study relates directly to mechanisms regulating the mineralization process of skeletal tissues and investigates the role of a protein which has been shown if mutated to lead to pathological or extensive mineralization in cartilage and bone. Therefore, this proposal might provide a novel therapeutic target to prevent pathological mineralization.